Owing to their specificity and high sensitivity, immunoassays are widely used in everyday clinical practice as well as in a variety of other applications such as environmental pollutant analysis and food quality analysis. Such assays are based on a specific binding between an antibody and an antigen.
Immunoassays based on electrochemical detection of binding have been a focus of considerable research and development activity (Ngo, T.T., Ed. 1987). In electrochemical immunoassay, what is detected is a change in electrical properties of an electrode solution interface as a result of antibody-antigen binding, such as changes in capacitance (Bresler, et al, 1992). Capacity affinity sensors have been disclosed in U.S. Pat. Nos., 4,728,822; 4,822,566 and 5,057,430, U.S. Pat. No. 4,893,957 and Canadian Patent Nos. 1,256,944 and 1,259,374. Other electrochemical immunoassay methods are based on a change in the current or potential response as a result of formation of antigen-antibody complexes. Such methods require the presence of a redox probe attached to the antibody or antigen and the response of the electrochemical assay is a result of competitive binding between the analyte and the redox labeled antibody or antigen with the complementary member pair immobilized on the electrode. Such methods are not very sensitive and the amperometric detection of the redox-labeled molcule is limited to the micromolar range (Weber, et al., 1979; Doyle et al., 1987). More recent work has shown that by using a bare electrode and coupling the redox reaction with an enzymatic amplification system (Di Gleria et al, 1986; Di Gleria et al, 1988) there is improvement of the sensitivity. Use of a similar approach, but with an electrode modified with a redox polymer, resulted in an immunoassay that was more sensitive (Chambers, et al, 1988).
Another approach includes direct covalent coupling of antigen or antibody with biocatalytic molecules (usually redox enzymes) for amplification of the electrochemical signal. A number of studies have utilized amperometric or potentiometric detection of electro-active species such as NADPH (Wright, et al, 1987; Tang, et al, 1991), phenol (Wehmeyer, et al, 1986), O.sub.2 (Aizawa, et al, 1987; Franconi, et al., 1987), H.sub.2 O.sub.2 (Uditha, et al., 1985; Tsuji, et al, 1990), NH.sub.3 (Gebauer, 1987), etc., generated analytically by an enzyme label on an antigen or antibody. The high sensitivity of this method makes it competitive with that of radioimmunoassay.
However, an electrical technique without any labeling of antigen/antibody species is even more attractive. Such methods are based on an amperometric detection of the permeability of redox molecules through a monolayer immobilized onto the surface of the electrode. An antigen monolayer provides much higher permeability to a solubilized redox probe than the same electrode reaction with an antibody with high binding affinity to the antigen (for illustration see FIG. 1) (Niwa, et al., 1992; Willner, et al, 1993; Willner et al, 1994; European Patent Application No. 668502).